Role of Peyer’s patches in the inductionof Helicobacter pylori-induced gastritisShigenori Nagai*†, Hitomi Mimuro†‡, Taketo Yamada§, Yukiko Baba*†, Kazuyo Moro*, Tomonori Nochi†¶,Hiroshi Kiyono†¶, Toshihiko Suzuki†‡, Chihiro Sasakawa†‡, and Shigeo Koyasu*†�

Departments of *Microbiology and Immunology and §Pathology, Keio University School of Medicine, Tokyo 160-8582, Japan; Divisions of ‡BacterialInfection and ¶Mucosal Immunology, Department of Microbiology and Immunology, Institute of Medical Science, University of Tokyo, Tokyo 108-8639,Japan; and †Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi, Saitama 332-0012, Japan

Edited by Jeffrey I. Gordon, Washington University School of Medicine, St. Louis, MO, and approved April 12, 2007 (received for review October 12, 2006)

Helicobacter pylori is a Gram-negative spiral bacterium that causesgastritis and peptic ulcer and has been implicated in the patho-genesis of gastric adenocarcinoma and mucosa-associated lym-phoid tissue lymphoma. Although Th1 immunity is involved ingastritis and the accumulation of H. pylori-specific CD4� T cells inthe H. pylori-infected gastric mucosa in human patients, how T cellsare primed with H. pylori antigens is unknown because no appar-ent lymphoid tissues are present in the stomach. We demonstratehere that Peyer’s patches (PPs) in the small intestine play criticalroles in H. pylori-induced gastritis; no gastritis is induced in H.pylori-infected mice lacking PPs. We also observed that the coccoidform of H. pylori is phagocytosed by dendritic cells in PPs. Wepropose that H. pylori converts to the coccoid form in the anaerobicsmall intestine and stimulates the host immune system through PPs.

CD4 T cells � coccoid form � dendritic cells � gastric epithelial cells �inflammation

Helicobacter pylori is a Gram-negative microaerophilic bac-terium that infects human gastric epithelial cell (gEC)

surfaces and the overlying gastric mucin. More than 50% of theworld’s population is infected by H. pylori, although most pa-tients have no remarkable symptoms (1). However, in some ofpatients, H. pylori infection leads to active chronic gastritis orpeptic ulcer (2). In addition, H. pylori has also been implicatedin the pathogenesis of gastric adenocarcinoma and mucosa-associated lymphoid tissue lymphoma (3). When H. pylori col-onizes gastric mucosa, effector molecules are injected intogastric epithelial cells or the submucosal area through the typeIV secretion system (1, 4). For example, the CagA effector isphosphorylated in the target cells and activates a signalingpathway to elicit growth factor-like responses. Another effectormolecule, VacA, causes the massive vacuolar degradation ofepithelial cells, thus disrupting the gastric epithelial barrier.VacA also interferes with the activation and proliferation of Tlymphocytes within the gastric lamina propria (gLP) (5).

It was originally proposed that effector molecules, includingCagA, trigger the secretion of chemokines such as IL-8 andRANTES from gECs, which attract neutrophils and mononu-clear cells into the gLP (4). However, it was later shown that H.pylori did not induce gastritis in lymphopenic SCID mice,although gastritis was induced after adoptive transfer of naiveCD4� T cells (6). The importance of CD4� T cells was under-scored by the fact that H. pylori is not eliminated from gastricmucosa in MHC class II-deficient mice (7).

Gastritis is more severe in Th1-prone mice than Th2-pronemice on infection with the mouse-adapted H. pylori strain SS1(8). Furthermore, the accumulation of H. pylori-specific CD4� Tcells in the H. pylori-infected gastric mucosa in human patients(9) suggests that CD4� T cell-mediated Th1 immune responsesplay a critical role in H. pylori-induced gastritis. However, howCD4� T cells are primed by H. pylori antigens in the stomachwhere no apparent lymphoid tissues are present and how the

H. pylori-induced chronic inflammation is maintained by T cellsis unknown.

Although H. pylori forms an actively dividing, spiral-shapedmorphology in the stomach, it is able to convert to a noncul-turable, but viable, coccoid form under unfavorable conditionssuch as an anaerobic environment, increased oxygen tension, andlong-term culture (10, 11). The coccoid form is thought to beimportant for transmission to new hosts by an oral–oral ororal–feces route because this form is more resistant to environ-mental stresses. Although the coccoid form is not culturable invitro, transcription and translation actively take place in thecoccoid form (12, 13). However, it is unknown whether thecoccoid form is involved in the pathogenesis of H. pylori-inducedgastritis.

In this study, we demonstrate that H. pylori antigen-specificCD4� T cells are necessary and sufficient for the induction ofgastritis by H. pylori. We also demonstrate that CD4� T cells arelikely primed with H. pylori antigens captured in the smallintestine, where the coccoid form of H. pylori is taken up bydendritic cells (DCs) in Peyer’s patches (PPs).

ResultsAdoptive Transfer of Naive CD4�T Cells Induces Gastritis in H. pylori-Infected Rag2�/� Mice. The H. pylori SS1 strain induces moresevere gastritis in Th1-prone C57BL/6 than Th2-prone BALB/cmice as demonstrated by the infiltration of neutrophils andlymphocytes into the gLP and the submucosal area (Fig. 1a anddata not shown). In contrast, when C57BL/6-Rag2�/� micelacking T and B cells were infected with H. pylori, no gastritis wasobserved (Fig. 1b), as previously shown with SCID mice (6). Theclearance of bacteria in Rag2�/� mice was impaired because�107 cfu/g tissues of H. pylori colonized the gastric mucosa(Table 1), and the colonization of H. pylori was readily detectedby anti-H. pylori antibody staining (Fig. 1c). However, adoptivetransfer of naive splenic CD4� T cells into Rag2�/� mice 2months after H. pylori infection induced severe gastritis, withmassive infiltration of neutrophils and lymphocytes into the gLPand the submucosal area (Fig. 1d). This massive infiltrationresulted in the exclusion of colonized H. pylori from gastricmucosa (Table 1).

Author contributions: S.N., H.K., C.S., and S.K. designed research; S.N., H.M., T.Y., Y.B., K.M.,T.N., and T.S. performed research; and S.N., C.S., and S.K. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Abbreviations: DKO, double knockout; LP, lamina propria; gLP, gastric LP; gEC, gastricepithelial cell; DC, dendritic cell; PPs, Peyer’s patches; OVA, ovalbumin; NK, natural killer;APC, antigen-presenting cell; �-Rag DKO, IL-2 receptor � chain (IL-2R�)�/�Rag2�/� DKO;�c-Rag DKO, cytokine receptor common � chain (�c)�/�Rag2�/� DKO; GALT, gut-associatedlymphoid tissue; ILF, isolated lymphoid follicle; BMDC, bone marrow-derived cell; SED,subepithelial dome; mLN, mesenteric lymph node.

�To whom correspondence should be addressed. E-mail: koyasu@sc.itc.keio.ac.jp.

This article contains supporting information online at www.pnas.org/cgi/content/full/0609014104/DC1.

© 2007 by The National Academy of Sciences of the USA

www.pnas.org�cgi�doi�10.1073�pnas.0609014104 PNAS � May 22, 2007 � vol. 104 � no. 21 � 8971–8976

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H. pylori Antigen-Specific CD4� T Cells Are Indispensable for Inductionof Gastritis. Primary gECs secrete MIP-2, a functional homologof IL-8, on H. pylori infection in vitro, and the amount producedby Rag2�/� gECs was comparable to that produced by wild-typegECs [supporting information (SI) Fig. 5a]. CD4� T cellsisolated from the gLP of H. pylori-infected mice were able toproduce larger amounts of MIP-2 than splenic CD4� T cellsfrom the same mice in response to H. pylori antigens (SI Fig. 5b).Moreover, the amounts of MIP-2 produced by gLP CD4� T cellswere much larger than those produced by gECs (compare SI Fig.5 a and b). The importance of CD4� T cells for neutrophilinfiltration on H. pylori infection was further confirmed by thedepletion of CD4� T cells from wild-type mice that had alreadydeveloped gastritis by H. pylori infection. After depleting CD4�

T cells by the i.v. injection of anti-CD4 mAb, the gastritis becamemilder (Table 1 and SI Fig. 6 a and b), and the number of bacteriain the gastric mucosa increased (Table 1). These results indicatethe critical role of CD4� T cells for both triggering and main-taining gastritis. When CD4� T cells from OT-II transgenic mice

on a Rag2�/� background (OT-II-Rag mice), specific for anovalbumin (OVA323–339 peptide on an MHC class II moleculeI-Ab), were transferred into H. pylori-infected Rag2�/� mice, nogastritis was induced (SI Fig. 6c). Similarly, when OT-II-Ragmice were infected with H. pylori, no gastritis was induced despitethe presence of CD4� T cells (Table 1 and SI Fig. 6d). Further-more, when OVA protein or OVA323–339 peptide was adminis-tered into H. pylori-infected OT-II-Rag mice, no inflammationwas observed, although CD4� T cells were activated in thesemice (Table 1, SI Fig. 6e, and data not shown). These resultscollectively indicate the importance of H. pylori antigen recog-nition by CD4� T cells in the induction of gastritis.

CD4� T Cells Are Not Primed with H. pylori Antigen in �c-Rag DKO Mice.IFN�, a key cytokine for Th1 immune responses, is important forthe pathogenesis of H. pylori-induced gastritis (14). Natural killer(NK) cells and antigen-presenting cells (APCs) including DCsare able to produce IFN� to prevent bacterial infection (15). Theinteraction between DCs and NK cells enhances the productionof IFN� during H. pylori infection (16, 17). To test the impor-tance of DC–NK interaction in the H. pylori-induced inflamma-tory response, we transferred splenic CD4� T cells intoH. pylori-infected IL-2 receptor � chain (IL-2R�)�/�Rag2�/�

(�-Rag DKO) mice and cytokine receptor common � chain(�c)�/�Rag2�/� DKO (�c-Rag DKO) mice. These mice lack NKcells because of impaired IL-15 signaling, which is critical for NKcell development (18, 19). In addition, the production of IL-12and IFN� by APCs from these mice is impaired (20). As shownin Fig. 1 e and f, gastritis was induced in H. pylori-infected �-RagDKO mice when naive CD4� T cells were transferred. Clearanceof bacteria was also achieved by the naive CD4� T cell transfer(Table 1), indicating that NK cells and NK–DC interaction aredispensable for the induction of gastritis by H. pylori infection.

Surprisingly, there was no gastritis induced in �c-Rag DKOmice even after the transfer of naive CD4� T cells (Fig. 1g), NKcells (SI Fig. 7a), or NK cells with naive CD4�T cells (SI Fig. 7b),suggesting that �c-Rag DKO mice have additional defects com-pared with �-Rag DKO mice. Interestingly, when splenic CD4�

T cells isolated from H. pylori-infected wild-type mice weretransferred, gastritis was induced in H. pylori-infected �c-RagDKO mice (Fig. 1h), and the clearance of bacteria was evident(Table 1). These results suggest that CD4� T cells were notprimed in �c-Rag DKO mice. In fact, splenocytes from thesemice did not respond to DCs preincubated with H. pylori lysate,whereas splenocytes from wild-type mice infected with H. pyloristrongly responded and produced IFN� in response to the sameDC preparation (data not shown). It should be noted that therewere no apparent defects in DCs from �c-Rag DKO micecompared with those from wild-type mice with regard to theirability to induce T cell activation and present antigen as exam-ined by the induction of CD69 expression and IFN� productionby splenic CD4� T cells (SI Fig. 8).

PPs Are a Critical Tissue for Priming CD4�T Cells with H. pylori Antigen.One difference between �-Rag DKO and �c-Rag DKO mice isthat the latter lack gut-associated lymphoid tissues (GALT) suchas PPs and isolated lymphoid follicles (ILFs) due to impairedIL-7 signaling (21) (Fig. 2 a–d). Thus, we hypothesized thatCD4� T cells are primed in GALT such as PPs or ILFs. To testthis possibility, we generated PP-null mice by administration ofanti-IL-7R� mAb in utero (22) (Fig. 2 e and f ). As observed in�c-Rag DKO mice, no gastritis was induced in PP-null mice 2months after H. pylori infection, and a large number of H. pyloriwas detected in the gastric mucosa (Fig. 2 h and i and Table 1).We also generated PP-null mice on a Rag2�/� background(PP-null-Rag2�/� mice) (Fig. 2g). The adoptive transfer of CD4�

T cells from H. pylori-infected wild-type mice, but not naiveCD4� T cells, induced strong inflammation in PP-null-Rag2�/�

Fig. 1. Naive CD4� T cells did not induce gastritis in H. pylori-infected �c-Ragdouble knockout (DKO) mice. (a–c) Wild-type (a) or Rag2�/� (b and c) micewere infected with H. pylori. Two months after the infection, gastric speci-mens were prepared. (d–h) Rag2�/� (d), �-Rag DKO (e and f ), and �c-Rag DKO(g and h) mice were infected with H. pylori. Two months after the infection,naive (d–g) or primed (h) splenic CD4� T cells were transferred. Two monthsafter the cell transfer, gastric specimens were prepared. Specimens werestained with H&E (a, b, d, e, g, and h), anti-H. pylori antisera (brown) (c), orchloroacetate esterase (red) for infiltrated neutrophils and mast cells ( f).(Scale bars: 200 �m.)

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mice just as in the �c-Rag DKO mice (Fig. 2 j and k and Table1). These results strongly suggest that PPs are critical for primingCD4� T cells in H. pylori infection, but dispensable for theeffector phase.

The Coccoid, but Not Helical, Form of H. pylori Is Phagocytosed by DCsin PP. Although H. pylori is helical in the stomach, it transformsto the coccoid form under anaerobic conditions, such as in thesmall intestine (23). Interestingly, the coccoid form of H. pyloriinduced higher IL-12 production from bone marrow-derivedcells (BMDCs) than the helical form (SI Fig. 9). There is apossibility that H. pylori transforms to the coccoid form in theintestine and is then captured by DCs present in PPs to inducea Th1 response. To test this possibility, the coccoid and helicalforms of H. pylori were inoculated into ligated small intestinalloops. As shown in Fig. 3a, immunofluorescence staining de-tected H. pylori in the subepithelial dome (SED) region of PPsin a time-dependent manner, and the number of bacteria in theSED region inoculated with the coccoid form was larger thanthat inoculated with the helical form of H. pylori. In addition,double immunofluorescence staining with anti-H. pylori anti-body and anti-CD11c mAb demonstrated that the bacteria werecaptured by CD11c� DCs in the SED region (Fig. 3b). Althoughthe helical form of H. pylori kept the rod shape 1.5 h afterinoculation (SI Fig. 10 a and b), the bacteria phagocytosed byCD11c� DCs in PPs were round (SI Fig. 10 c and d). Theseresults suggest that the coccoid, but not the helical, form ofH. pylori is captured by DCs in PPs and activates immuneresponses by generating H. pylori-specific pathogenic CD4� Tcells. Consistent with this observation, CD4� T cells from thePPs as well as mesenteric lymph node (mLN) of H. pylori-infected wild-type mice were also able to eliminate the bacteriain �c-Rag DKO mice infected with H. pylori (Table 1).

When wild-type mice were infected with the coccoid form ofH. pylori, gastritis was not induced because the coccoid form ofH. pylori was unable to colonize in the stomach (Fig. 4a and datanot shown). However, CD4� T cells from these mice inducedgastritis in �c-Rag DKO mice infected with the helical form ofH. pylori (Fig. 4 b and c and Table 1). These results indicate thatCD4� T cells primed with the coccoid form of H. pylori in the

intestine are sufficient to induce inflammation in the stomachinfected with the helical form of H. pylori.

DiscussionWe showed here that H. pylori antigen-specific CD4� T cells arerequired to induce and maintain gastritis on infection withH. pylori. Because H. pylori interacts with and injects pathologicalmolecules into gECs, it is generally thought that neutrophilsinfiltrating the gLP are attracted by chemokines produced bygECs. However, neutrophil infiltration was not observed inRag2�/� mice, although gECs of Rag2�/� mice were able tosecret MIP-2 on H. pylori infection. Thus, the secretion ofchemokines by gECs seems insufficient for the induction ofgastritis. In addition, adoptive transfer of CD4� T cells recog-nizing H. pylori-independent antigens did not induce gastritis,suggesting that H. pylori-specific CD4� T cells directly or indi-rectly regulate production of chemokines that attract neutro-phils. In fact, a large amount of MIP-2 was produced by activatedCD4� T cells derived from the gLP of H. pylori-infected mice. Inaddition, infiltrated neutrophils were located around CD4� Tcells in the gLP of H. pylori-infected mice (data not shown). Itis known that another keratinocyte-derived chemokine is able torecruit neutrophils. However, the amounts of keratinocyte-derived chemokine produced by both gECs and CD4 T cells weremuch lower than those of MIP-2 (data not shown).

Oral or intra-PP immunization with H. pylori antigens waseffective in enhancing H. pylori-specific CD4� T cell responsesand reducing H. pylori colonization in the stomach (24, 25).These reports are consistent with our current observation thatPPs play critical roles in priming CD4� T cells, and H. pylori isindeed captured by DCs in PPs. H. pylori antigen-specific CD4�

T cells would be primed by DCs in PPs or mLNs where DCsmigrate after capturing antigens. Interestingly, CD4� T cellscannot be primed by DCs in the gLP or gEC, both of which arecapable of expressing class II MHC and presenting antigens. Thelack of antigen presentation is partly due to the fact that thehelical form of H. pylori is resistant to phagocytosis in a type IVsecretion system-dependent manner, although the molecularmechanisms of antiphagocytic activity remain to be determined(26). It should be noted that the transformation of the helical tothe coccoid form is accompanied by changes in the composition

Table 1. PP-dependent bacterial clearance in H. pylori infection

Mouse* n Cells transferred†

Bacterialcolonization,‡

cfu/g tissue � 10�6

Neutrophils,average (range)

Activeinflammation,

average (range)GAIS,§

average (range)

Wild type 7 None 2.2 � 1.3 1.6 (0–3) 1.4 (0–3) 13.6 (0–34)Wild type 4 CD4� T cell-depleted 25 � 7 0 (0) 1.0 (0–2) 0 (0)Rag2�/� 4 None 14 � 4 0 (0) 0 (0) 0 (0)Rag2�/� 5 Naive CD4� T 0.15 � 0.17 2.0 (2) 2.0 (2) 5.0 (2–8)Rag2�/� 5 OT-II-Rag CD4� T 34 � 11 0 (0) 0 (0) 0 (0)�-Rag DKO 4 Naive CD4� T 0.67 � 0.39 1.5 (1–2) 1.3 (1–2) 9.2 (3–18)�c-Rag DKO 6 Naive CD4� T 18 � 11 0 (0) 0 (0) 0 (0)�c-Rag DKO 6 Primed CD4� T 0.22 � 0.39 0.66 (0–1) 1.5 (0–2) 1.6 (0–5)�c-Rag DKO 3 Primed CD4� T from PPs �0.01 1.0 (1) 2.0 (2) 1.0 (0–2)�c-Rag DKO 3 Primed CD4� T from mLN 0.88 � 0.78 0.5 (0–1) 1.5 (1–2) 4.0 (3–5)�c-Rag DKO 3 Primed CD4� T by coccoid form 1.8 � 1.6 0.33 (0–1) 0.66 (0–1) 2.0 (0–6)PP-null-wild type 8 None 16 � 9 0 (0) 0.37 (0–1) 0 (0)PP-null-Rag2�/� 3 Naive CD4� T 5.3 � 4.0 1.0 (0–2) 1.0 (0–2) 4 (0–7)PP-null-Rag2�/� 3 Primed CD4� T 0.53 � 0.28 2.6 (2–3) 2.6 (2–3) 21 (13–33)

*All mice were on a C57BL/6 background. Although not shown, the degrees of bacterial colonization in �-Rag DKO, �c-Rag DKO, and PP-null-Rag2�/� micewithout CD4� T cell transfer were similar to those of Rag2�/� mice.

†Splenocytes were used for cell transfer unless otherwise stated. Five million cells were transferred except for the transfer of PP-derived cells, where 5 � 105 cellswere used.‡Mean � SD.§Gland active inflammatory score. See Materials and Methods.

Nagai et al. PNAS � May 22, 2007 � vol. 104 � no. 21 � 8973

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of surface proteins and/or carbohydrates, which may make thebacteria susceptible to phagocytosis (27, 28), a subject worthy offurther studies. It has been shown that mast cells are able topresent H. pylori antigens to H. pylori-specific CD4� T cells,which in turn activate mast cells to degranulate (29). When weinfected W/Wv and Sl/Sld mice lacking mast cells with H. pylori,gastritis was readily induced in both strains of mice on infection(S.N., T.Y., Y.B., and S.K., unpublished data), indicating thatmast cells are not essential in priming CD4� T cells. In an in vitroexperiment, BMDCs infected with the coccoid form of H. pyloriproduced larger amounts of IL-12 than those infected with thehelical form of H. pylori, suggesting that the coccoid form of

H. pylori easily induces Th1 immune responses on H. pyloriinfection.

Importantly, CD4� T cells primed with the coccoid form ofH. pylori were able to induce gastritis in H. pylori-infectedGALT-null �c-Rag DKO mice where CD4� T cells are not

Fig. 2. PPs are critical for the priming of CD4� T cells in H. pylori infection.(a–e) Small intestines from wild-type (WT) (a) and PP-null (e) mice were stainedwith anti-B220 mAb, and small intestines from Rag2�/� (b), �-Rag DKO (c), and�c-Rag DKO (d) mice were stained with anti-CD45 mAb. ( f and g) Schemes ofthe generation of PP-null WT ( f) or PP-null-Rag2�/� (g) mice. (h and i) PP-nullWT mice were infected with H. pylori. Two months after the infection, gastricspecimens were prepared. (j and k) PP-null-Rag2�/� mice were infected withH. pylori. Two months after the infection, naive (j) or primed (k) splenic CD4�

T cells were transferred. Two months after the cell transfer, gastric specimenswere prepared. Specimens were stained with H&E (h, j, and k) or anti-H. pyloriantisera (brown) (i). (Scale bars: 200 �m.)

Fig. 3. The coccoid form of H. pylori is captured by DCs in PPs. The coccoidor helical form of H. pylori was inoculated into the ligated small intestines ofwild-type mice. (a) After the indicated incubation times, PPs were stained withanti-H. pylori antibody. (b) Twenty hours after inoculation of the coccoidform, PPs were stained with anti-CD11c mAb (green) and anti-H. pyloriantibody (red). (Scale bars: 0 h,100 �m; 20 h, 20 �m.)

Fig. 4. Gastritis is induced by CD4� T cells primed by the coccoid form ofH. pylori. Two months after infection of �c-Rag DKO mice with the helical formof H. pylori, splenic CD4� T cells from wild-type (WT) mice orally infected bythe coccoid form of H. pylori were transferred to the infected �c-Rag DKO mice(a). Two months after the cell transfer, gastric specimens were prepared.Specimens were stained with H&E (b) or chloroacetate esterase (red) forinfiltrated neutrophils and mast cells (c). (Scale bars: 200 �m.)

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primed with H. pylori antigen at all. Thus, the following scenarioemerges from our results: H. pylori transforms to the coccoidform when entering the intestinal tract and is captured by DCsin PPs. H. pylori antigens presented by DCs are recognized byCD4� T cells in PPs or mLN, and activated T cells migrate to thegastric mucosa to induce and maintain inflammatory responses.We noted that PP-null-Rag2�/� mice exhibited modest inflam-mation in the stomach after naive CD4� T cell transfer, com-pared with �c-Rag DKO mice, which showed no sign of inflam-mation. Because treatment by anti-IL-7R� mAb in uterosuppresses PP development, but leaves ILFs and recently dis-covered villous M cells (30) intact, ILFs and/or villous M cellsmay also participate in the capture of H. pylori in the intestine.Although luminal antigens can be taken up by M cells locatedover the follicular epithelia of ILF, the tissue is predominantlyoccupied by B cells (31). Furthermore, in a separate study, wehave shown that no apparent organized lymphoid structure isdeveloped under villous M cells (data not shown).

H. pylori is also implicated in the cause of other diseases suchas idiopathic thrombocytopenic purpura (32) and Sjogren syn-drome (33). Indeed, it has been shown that T cells migrate fromthe intestine to the salivary gland in Sjogren syndrome patients(34). It will be of interest to examine the functional relationshipbetween these diseases and the coccoid form of H. pyloricaptured via PPs.

Materials and MethodsMice. All mice used in this study were on a C57BL/6 background andwere maintained at Taconic (Germantown, NY) or in our animalfacility under specific pathogen-free conditions. Wild-typeC57BL/6 mice were purchased from Sankyo Labo Service (Shi-zuoka, Japan) and CLEA Japan (Tokyo, Japan). Rag2�/� mice,�c-Rag DKO mice, and OT-II-Rag mice were obtained fromTaconic. IL-2R��/� mice (35) were generously provided by T. W.Mak (Ontario Cancer Institute, Toronto, ON, Canada). �-RagDKO mice were obtained by crossing IL-2R��/� with Rag2�/�

mice (20). All experiments were approved by the Animal Care andUse Committee of the Keio University School of Medicine andwere performed in accordance with institutional guidelines.

Antibodies. Fluorescein-conjugated antibodies for f low-cytometric analysis and biotin anti-mouse CD2c (HL3) werepurchased from BD Bioscience (San Jose, NJ). Anti-H. pyloriantibodies were purchased from Biomeda (Foster City, CA) orDAKO (Glostrup, Denmark).

Bacteria. H. pylori strain SS1, a mouse-adapted human isolate,was used for all experiments. To prepare the helical form ofH. pylori, SS1 was grown on 5% sheep blood agar plates for 2days. Before inoculating into mice, bacteria were grown inBrucella broth with 5% FCS overnight at 37°C under microaero-bic conditions with gentle agitation. To prepare the coccoidform, SS1 was grown on 5% sheep blood agar plates undermicroaerobic conditions for 3 days at 37°C and then culturedunder anaerobic conditions for 7 days at 37°C.

In Vivo Infection of Mice. Bacteria were prepared from logarithmicphase cultures. Mice were intragastrically infected with 1–2 � 108

cfu H. pylori in 0.15 ml of broth. After the indicated time period,mice were killed and the stomach was aseptically removed. Thestomach was then bisected along the greater and lesser curva-tures. Half of the stomach was homogenized for the determi-nation of bacterial colonization by a plate-dilution method. Therest of the stomach was sectioned transversely into two strips forfrozen and paraffin-embedded sections.

In Situ Infection of Intestinal Loop. Wild-type mice (6-week-oldfemales) were anesthetized by an i.m. injection of 2 mg of

ketamine hydrochloride (Sankyo, Tokyo, Japan) and 0.1 mg ofXylazine per mouse. An �4-cm-long piece of the small intestinecontaining one or two PPs was ligated at both ends with surgicalthread. H. pylori (1 � 109) suspended in 0.2 ml of saline wasinoculated into the loop. After the indicated time periods, PPswere removed and extensively washed with PBS. After fixationin 4% paraformaldehyde in PBS, specimens were processed forhistopathological examination.

Generation of PP-Null Mice. PPs were depleted from a smallintestine as previously described (22). Briefly, 14.5 days postco-itum pregnant wild-type or Rag2�/� mice were i.v. injected with2 mg of anti-IL-7R� mAb (A7R34; kindly provided by S.-I.Nishikawa, RIKEN CDB, Kobe, Japan). To confirm the deple-tion of PPs, a dissected small intestine from one of the offspringwas fixed with acetone and stained with anti-B220 or anti-panCD45 mAb (BD Biosciences).

Adoptive Transfer of CD4� T Lymphocytes. Naive or H. pylori antigen-primed CD4� T cells were purified from splenocytes, mLN, and PPsby using anti-mouse CD4-microbeads and AutoMACS (MiltenyiBiotech, Sunnyvale, CA) according to the manufacturer’s instruc-tion. The purity of isolated cells was �95%. Isolated cells (5 � 106

per mouse for splenocytes and mLN, 5 � 105 per mouse forPP-derived cells) were injected i.v. into recipient mice infected withH. pylori for 8 weeks. Eight weeks after the transfer, mice were killedfor the indicated analyses.

Histological Analysis. An excised stomach was fixed in a neutral-buffered 10% formalin solution and cut into four strips. Sampleswere processed by standard methods, embedded in paraffin, andsectioned at 4 to 5 �m. Specimens were stained with H&E orused for cytochemical and immunohistochemical studies. TheLeder method was used to assess naphtol-AS-D-chloroacetateesterase detection (36). Immunohistochemical analysis was per-formed with formalin-fixed and paraffin-embedded tissue sec-tions by using heat-induced epitope retrieval and the ABC(Vectastain ABC kit; Vector Laboratories, Burlingame, CA)method. Anti-H. pylori serum from DAKO was used for H. pyloristaining. In some cases, frozen sections (7 �m) were prepared,fixed with 4% paraformaldehyde in PBS, and blocked with 2%BSA-PBS, and immunofluorescence was performed using thetyramide amplication method (TSA-Plus Fluorescein System;PerkinElmer Life and Analytical Sciences, Boston, MA) andthen incubated with anti-H. pylori antibody from Biomeda,followed by Cy5 (GE Healthcare Bioscience AB, Uppsala,Sweden) or TRITC-linked rabbit IgG (Sigma–Aldrich, St. Louis,MO). The specimens were mounted with Vectashield (VectorLaboratories) and examined with a confocal laser-scanningmicroscope LSM510 by using version 3.2 software (Carl Zeiss,Thornwood, NY). The zymogenic zone of middle corpus �3 mmfrom the FS/Z transition zone was examined in each sample.

Histological Score. For assessment of gastric histopathology,blinded sections stained with H&E were examined by lightmicroscopy. Neutrophil infiltration was assessed by the presenceof neutrophils in the gastric mucosa. Active inflammation wasassessed by the degree and area of damages of mucosal tissue andmuscular layers because of infiltrations of neutrophils, lympho-cytes, and/or macrophages. The scoring was graded as 0 (no), 1(mild), 2 (moderate), or 3 (severe). The total number of glandswith neutrophil infiltration in the crypt and lumen was alsocounted to produce a gland active inflammatory score.

Whole-Mount Immunohistochemistry. Small intestines were removedand stained with antibodies as described previously (37). Briefly,small intestines were washed, incubated twice in HBSS containing5 mM EDTA at 37°C for 20 min, and fixed with ice-cold formalin

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for 1 h. After blocking, specimens were incubated with biotin-conjugated anti-B220 mAb for wild-type mice or anti-CD45 mAbfor mice on a Rag2�/� background in Solution A containing 0.6%Triton X-100 and 0.1% BSA for 1 h, incubated with ABC reagent(Vector Laboratories) at room temperature for 2 h, and reactedwith diaminobenzidene.

We thank Drs. S.-I. Nishikawa and T. W. Mak for valuable materials; Dr.L. K. Clayton for valuable discussion and critical reading of the manu-

script; and M. Motouchi and N. Yumoto for animal care. This work wassupported by the Waksman Foundation of Japan (S.N.), the UeharaMemorial Foundation, the Tokyo Biochemical Research Foundation,Japan Society for the Promotion of Science Grant-in-Aid 18659141,Scientific Research on Priority Areas Grant-in-Aid 14021110, a NationalGrant-in-Aid for the Establishment of a High-Tech Research Center ina Private University, a grant for the Promotion of the Advancement ofEducation and Research in Graduate Schools, and a Scientific FrontierResearch Grant from the Ministry of Education, Culture, Sports,Science, and Technology, Japan.

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